GSM7653207: WT B cells H3K27me3 ChIP DMSO Rep2; Mus musculus; ChIP-Seq
Harvested cells were washed with PBS and fixed with 1% formaldehyde/PBS for 5 min at RT. The reaction was quenched with 0.125 M glycine for 5 min at RT. Fixed cells were washed with PBS and lysed to isolate chromatin. Chromatin was sonicated with Bioruptor Pico (Diagenode) to < 500 bp. Sonicated chromatin was diluted ten-fold and pre-cleared with Dynabeads Protein G (ThermoFisher). A small volume (5%) was saved as input. The remainder was incubated with 4 ug anti-H3K27me3 antibody per 30 ug chromatin over night. Dynabeads Protein G was added and incubated for 2 hours. Immunoprecipitated chromatin-protein complexes were washed and decrosslinked over night at 65 C. Input and ChIP DNA were treated with RNaseA and proteinase K, column purified (NEB T1030S), and quantified with Qubit (ThermoFisher). Libraries were prepared with NEBNext Ultra II DNA kit (E7645S) and NEBNext Multiplex Oligos (E7600S), following the manufacturer's recommendations. Size selection and purification were performed with Ampure XP beads (Beckman Coulter).